The present invention relates to a system for the regulation of gene expression. The invention provides improved nucleic acid constructs capable of tightly regulating the expression of a coding sequence of interest. Tight regulatory control is desirable where the nucleic acid to be expressed, or the level of expression, is toxic to the cellular or host environment in which expression occurs. The invention also relates to the application of the expression system in cells used to package viral vectors and provides methods of preparing the necessary nucleic acid constructs as well as their use in the control of recombinant viral gene expression. In one aspect, the invention relates to the regulation of gene expression in a stably transfected cell.
Recombinant nucleic acid technology has proven to be a powerful tool for the expression of the products encoded by nucleic acids of interest. This has resulted in the ability to produce polypeptides and nucleic acids for both research and commercial applications.
Some encoded products, however, are toxic to the cellular or host environment in which their expression occurs, either because the product is inherently toxic or because the levels at which expression occurs is so high as to result in toxicity. One means of dealing with this difficulty has been to use transient expression systems wherein the encoded product is expressed and recovered before toxicity results in reduced levels of product. Alternatively, the encoded product is placed under a tightly controlled regulation system such that the product may be expressed and then expression terminated before toxicity rises to lethal levels. One example of a tightly controlled regulatory system is seen with the use of a tetracycline regulated operator/promoter in combination with a tet repressor (see for example U.S. Pat. No. 5,750,396).
The expression of a toxic product is of particular importance in situations where the product must be continually expressed because it is a component of a larger product being produced, or metabolic activity being conducted, by the cell. One example of such a situation is in the case of a viral packaging cell line, which expresses products necessary for the assembly and packaging of viral particles. If any one of the necessary viral gene products is toxic to the cell, the need to control its expression becomes critical if a stable (as opposed to transient) packaging cell line is to be used. One example of a necessary toxic viral gene is in the case of the G protein from vesicular stomatitis virus (VSV), which is desirable for the production of pseudotyped viral particles.
An example using the tet operator and repressor to regulate the expression of VSV-G is described by Henriette et al. (J. Virol. 73(1):576-584, 1999), where the tet repressor (as a chimeric fusion product with a domain of VP-16 and referred to as tTA) is under the control of a cytomegalovirus (CMV) promoter and VSV-G is under the control of a tet operator. Expression of the chimeric repressor in the absence of tetracycline results in no expression of VSV-G. The presence of tetracycline prevents association between tTA and the tet responsive elements (TRE) found in the operator to allow the expression of VSV-G. This system is referred to as xe2x80x9ctet-onxe2x80x9d where the presence of tetracycline results in the expression of the gene of interest (i.e. VSV-G).
There is also an alternative xe2x80x9ctet-offxe2x80x9d system where tTA is a chimeric transactivator. It cannot bind to the TRE of a tet operator in the presence of tetracycline. But in the absence of tetracycline, tTA binds to the operator and strongly activates the promoter to express a coding sequence of interest.
Klages et al. (Molec. Therap. 2(2):170, 2000) teach the use of a similar two nucleic acid system to control VSV-G expression. The first nucleic acid expresses tTA which then controls a TRE containing tet operator that controls VSV-G expression. The same tTA protein also regulates expression of the rev protein which in turn regulates the expression of the gag and pol regions (necessary for viral packaging) by controlling the splicing of the gag/pol messenger RNA via a rev responsive element (RRE).
Another example of the use of the rev protein to control gene expression was described by Yu et al. (J. Virol. 70(7):4530-4537, 1996). They used the expression of tTA to regulate the expression of both HIV-1 rev and envelope proteins which were simultaneously under the regulation of a single TRE containing tet operator. The rev protein then in turn regulates expression of the viral envelope protein, via an RRE, as well as the expression of the gag/pol messenger RNA via another RRE. While transcription of the gag/pol coding sequences was regulated by another promoter, its expression was directly regulated by the rev protein and thus indirectly regulated by tTA.
The present invention provides nucleic acid (expression) constructs and methods for regulating the expression of one or more than one coding sequence of interest. The nucleic acid constructs are preferably recombinant in nature and include at least three constructs where the last one contains the coding sequence of interest. The constructs may be viewed and used as an expression system to express the coding sequence of interest, where each construct express a product that regulates the expression of the next construct in turn so that ultimately, expression of the coding sequence of interest via the last construct is controlled. Each construct preferably, and individually, contains a regulatory region, such as a promoter (optionally with an operator).
In one aspect of the invention, the expression system is utilized as part of a cell or cell line, used to package viral vectors, to regulate expression of components needed to package the vector. The expression system may be used to regulate expression of viral structural or regulatory gene products necessary for packaging a viral vector of interest. Where more than one viral gene product of interest is to be expressed, they may be located on separate nucleic acid molecules and still remain part of the expression system of the present invention. The expression systems of the invention may thus have constructs in common such that two or more systems may be combined to express two or more coding sequences of interest regulated by said systems. Examples of systems with constructs in common include the use of the same first and second nucleic acid constructs but with two third constructs containing two coding sequences of interest, both of which are regulated by the same mechanism via the first and second nucleic acid constructs.
In another aspect of the invention, expression from the first nucleic acid construct is preferably tightly regulated or even autoregulated. One non-limiting example is through a positive feedback mechanism where the product of the first nucleic acid construct can repress its own expression. In the absence of activation, this autoregulation of the first nucleic acid construct allows for a very low basal activity such that little to no expression of the coding sequences of interest (in a additional nucleic acid construct) occurs. Once expression of the first nucleic acid construct is activated, the expression of all additional constructs in the system follows. Autoregulation of the first construct is used in preferred embodiments of the invention to maximize control of expression from the additional constructs in the system.
The constructs and systems of the invention may be incorporated into vectors or introduced into cells. With cells, the constructs may be integrated into the cellular genome or maintained as episomal constructs. The choice of cell is not critical so long as it is permissive for the expression of the constructs and systems of the invention. In embodiments of the invention wherein the cells are used to package viral vectors, the resultant viral vector is preferably complement resistant.
Each construct of the invention may be present on an individual nucleic acid molecule or present on the same nucleic acid molecule as one or more than one of the other constructs (for example, but not limited to, the same plasmid, vector, or chromosome). In preferred embodiments of the invention where the expression system is used in cells to package viral vectors, the individual constructs are preferably divided into separate nucleic acid molecules. The presence of the constructs on one or more than one nucleic acid molecule is generally not critical to the practice of the invention so long as the arrangement of constructs does not result in interference of each construct""s ability to regulate the expression of any subsequent construct.
Alternatively, and in an additional aspect of the invention, the constructs are positioned such that there is a regulatory effect arising from the arrangement. A non-limiting example is where two constructs are positioned on one molecule so that their promoters are oriented to express sequences divergently (where the 5xe2x80x2 portions of each promoter are closer together than the 3xe2x80x2 portions of each promoter) so that activation of one promoter increases the ease of activating the other. This arrangement on plasmids, when used in cells to package viral vectors, has been found to increase the titer of vector production. The arrangement may be mirrored in cells containing a stable integration of nucleic acid constructs by preparing two constructs to be divergently oriented on a single molecule and then integrating the molecule into cellular genome. The divergent orientation would thus be maintained along with the relative positions of the two constructs.
In a further aspect of the invention, one or more than one product expressed by one or more than one construct of the invention is preferably viral in origin and capable of stringently controlling expression from another construct. A preferred expression system of the invention thus expresses one or more than one viral regulatory protein which can regulate expression from another construct of the system.
The constructs and systems of the invention are preferably used to regulate the expression of sequences coding for a product or products toxic to a cell or host, such as, but not limited to, viral proteins. The constructs and systems may also, of course, be used to regulate the expression of non-toxic products.
In one preferred aspect of the invention, the constructs of the invention are incorporated into cells used to package viral vectors into viral particles. Such cells may be referred to as xe2x80x9cpackaging cellsxe2x80x9d because they produce all the necessary components to package viral vector nucleic acids into viral particles. Recombinant viral nucleic acids, optionally containing other heterologous or endogenous sequences of interest, may be packaged by such cells. In aspects of the invention where the nucleic acid constructs result in an inducible system to regulate expression of the necessary viral components, such cells may also be viewed and used as an inducible system for packaging viral particles.
The constructs of the invention may be introduced into cells to result in the maintenance of the constructs as extrachromosomal and/or integrated copies. In preferred embodiments of the invention, integration of the constructs is used to produce a stably transfected cell line that may be used to package viral particles for an extended or indefinite period of time.
The invention also provides methods for the production of the constructs and systems described above as well as methods for the use of such systems to regulate expression of coding sequences. These methods include the use of the invention to produce one or more than one component necessary for packaging a viral vector. Kits containing the constructs of the invention or for use with the disclosed methods are also provided.